This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. RNA polymerase is one of the largest protein complexes in the cell and is responsible for transcribing RNA using DNA as templates. Although the core complex can bind non-specific DNA, it requires an appropriate sigma factor to form the holoenzyme for specific promoter binding and transcription initiation. Upon binding to a promoter, the sigma factor facilitates melting of the downstream DNA to form the transcription initiation bubble, and thus converting the RNA polymerase to an open complex competent for initiating RNA synthesis. The goal of this project is to capture the individual steps of transcription initiation. The holoenzyme we are working on is the E. coli RNA polymerase with bound primary sigma factor (sigma-70).